Toxoplasma gondii is an intracellular protozoan parasite that represents a major threat to immunocompromised individuals, including AIDS patients, newborns with a congenital infection, and organ transplant recipients. Toxoplasma is a permanent infection that evades the host immune response because the acute form (tachyzoite) can develop into a latent cyst form (bradyzoite) in response to stress. We wish to address the mechanisms by which Toxoplasma detects cellular stresses and triggers differentiation into the cyst form. A well characterized stress response pathway conserved in eukaryotic cells involves translational control by virtue of the phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF21). When eIF21 is phosphorylated at its regulatory serine (serine-51), global translation initiation is dampened, decreasing the synthesis of the current proteome so the cell can conserve energy and reprogram gene transcription to remedy the stress. We have demonstrated that the eIF21 homolog in Toxoplasma (TgIF21) is phosphorylated in response to a variety of cellular stresses, including stress conditions that induce cyst formation. Additionally, TgIF21 remains highly phosphorylated in bradyzoite cyst forms that are induced in vitro, and pharmacological inhibition of eIF21 dephosphorylation induced the expression of bradyzoite specific genes and promoted mature cyst formation in vitro. We recently made transgenic parasites that cannot phosphorylate the regulatory serine reside (Ser71) of TgIF21 in a type I (RH) strain. The RH TgIF21-S71A mutant parasites demonstrated that TgIF21 phosphorylation is critical for viability because they are ill-equipped to cope with exposure to the extracellular environment upon lysis of the host cell. Therefore, we hypothesize that TgIF21 kinases are critical for persistence of both acute and chronic infections by allowing Toxoplasma to adapt to environmental stresses. To address this hypothesis, in AIM 1 we will systematically create knockouts for each of the four Toxoplasma eIF21 kinases (TgIF2K-A through D) in a type I background. These novel mutants will allow us to dissect the function of each eIF21 kinase in responding to a diverse set of cellular stresses in tachyzoites, with an eye for future drug targets. In Aim 2, we will create a type II parasite strain that is incapable of TgIF21 phosphorylation by converting the regulatory Ser71 to Ala. The type II TgIF21-S71A will establish the role of stress-induced TgIF21 phosphorylation on Toxoplasma development and cyst latency. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a protozoan parasite that causes significant disease as an opportunistic infection of AIDS patients. Toxoplasma is a permanent infection that evades the host immune response because the proliferative form can develop into a latent cyst form in response to cellular stress. The mechanisms that govern acute stress response and stress-induced cyst formation will identify novel points of therapeutic intervention. We are taking an innovative approach to study translational control as a means to elucidate the mechanisms of acute infection and microbial latency. Knowledge of these stress directed pathways will provide potential new therapeutic targets for the treatment of Toxoplasma infections and related pathologies in AIDS patients.